LOCUS       sequence-62777-e         931 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  natural linear DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 931)
  AUTHORS   Scholz J, Besir H, Strasser C, Suppmann S
  TITLE     A new method to customize protein expression vectors for fast, 
            efficient and background free parallel cloning.
  JOURNAL   BMC Biotechnol. 2013 Feb 14;13(1):12.
  PUBMED    23410102
REFERENCE   2  (bases 1 to 931)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC 
            Biotechnol."; date: "2013-02-14"; volume: "13(1)"; pages: "12"
COMMENT     Sequence Label: pCoofy33
FEATURES             Location/Qualifiers
     source          1..931
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     53..72
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        53..71
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    72..96
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             111..133
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             146..175
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             185..208
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     RBS             324..332
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             334..642
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MIQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSD
                     KVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     521..540
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             join(647..652,653..676,677..712,713..736)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             740..757
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(810..828)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      824..871
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa attaatacga
       61 ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac
      121 tttaagaagg agatatacca tgaaacatca tcatcatcat caccatcacc atcactccgc
      181 gggtctggaa gttctgttcc aggggcccat ggactaatga ggtaccggat ccgaattcga
      241 gctccgtcga caagcttgat atcgaattcc tgcagcccaa actgcagttg acaacataaa
      301 aactttgtgt tatacttgta acgtaaggag gtaatgattc agtttaaggt ttacacctat
      361 aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccc
      421 gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt
      481 gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg
      541 gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat
      601 gacatcaaaa acgccattaa cctgatgttc tggggaatat aagcttagcg cttggagcca
      661 cccgcagttc gaaaaaggtg gaggttctgg cggtggatcg ggaggttcag cgtggagcca
      721 cccgcagttc gagaaataac accaccacca ccaccactga gatccggctg ctaacaaagc
      781 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
      841 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
      901 gaatgggacg cgccctgtag cggcgcatta a
//
